BIOSENSORS & BIOELECTRONICS
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2025
·
IF 10.5
Junjie Zeng,Yanping Li,Qinghua He,Ji Xia,Jiebiao Guo,Zhui Tu
Phage display-mediated immuno-polymerase chain reaction (PD-IPCR) combines the strengths of phage display technology and immuno-PCR. The link between genotype and phenotype has been established by displaying antibody fragments on the surface of phage particles, allowing signal transmission. In this study, we utilized aflatoxin B 1 (AFB 1 ) as a model system to investigate the efficacy of PD-IPCR. Signal amplification was achieved using both real-time fluorescence quantitative PCR (qPCR) and a custom-made droplet digital PCR (dPCR) system. Under optimized conditions, the assay using dPCR achieved a limit of detection of 0.015 ng/mL, a 6-fold improvement over qPCR. The validation of our method using major agricultural produce, such as corn, flour, and soy sauce, shows excellent recoveries ranging from 85.77 % to 108.28 %, with a total assay time of less than 2 h. Furthermore, we compared our results with a high-performance liquid chromatography (HPLC) method for AFB 1 content in corn and flour, revealing consistent outcomes (r = 0.9847). These results demonstrate the potential of nanobody-based PD-IPCR as a reliable tool for detecting mycotoxins.
研究领域:
食品安全
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
BIOMEDICAL MICRODEVICES
·
2023
·Vol. 25
·期 2
·1-7
·
IF 2.8
sun Kaixin,Whiteside Ben,Hebda Michael,Fan Yiqiang,Zhang Yajun,Xie Yumeng,Liang KunMing
Polymerase chain reaction (PCR) has become a powerful tool for detecting various diseases due to its high sensitivity and specificity. However, the long thermocycling time and the bulky system have limited the application of PCR devices in Point-of-care testing. Herein, we have proposed an efficient, low-cost, and hand-hold PCR microdevice, mainly including a control module based on water-cooling technology and an amplification module fabricated by 3D printing. The whole device is tiny and can be easily hand-held with a size of about 110 mm × 100 mm × 40 mm and a weight of about 300 g at a low cost of about $170.83. Based on the water-cooling technology, the device can efficiently perform 30 thermal cycles within 46 min at a heating/cooling rate of 4.0/8.1 ℃/s. To test our instrument, plasmid DNA dilutions were amplified with this device; the results demonstrate successful nucleic acid amplification of the plasmid DNA and exhibit the promise of this device for Point-of-care testing.
研究领域:
生物医学工程
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分子诊断
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医疗器械
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产品关联
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2015
·
IF 6.723
Yalong Bai,Yan Cui,George C. Paoli,Chunlei Shi,Dapeng Wang,Xianming Shi
Nanomaterials have been widely reported to affect the polymerase chain reaction (PCR). However, many studies in which these effects were observed were not comprehensive, and many of the proposed mechanisms have been primarily speculative. In this work, we used amino-modified silica-coated magnetic nanoparticles (ASMNPs, which can be collected very easily using an external magnetic field) as a model and compared them with gold nanoparticles (AuNPs, which have been studied extensively) to reveal the mechanisms by which nanoparticles affect PCR. We found that nanoparticles affect PCR primarily by binding to PCR components: (1) inhibition, (2) specifity, and (3) efficiency and yield of PCR are impacted. (1) Excess nanomaterials inhibit PCR by adsorbing to DNA polymerase, Mg2+, oligonucleotide primers, or DNA templates. Nanoparticle surface-active groups are particularly important to this effect. (2, a) Nanomaterials do not inhibit nonspecific amplification products caused by false priming as previously surmised. It was shown that relatively low concentrations of nanoparticles inhibited the amplification of long amplicons, and increasing the amount of nanoparticles inhibited the amplification of short amplicons. This concentration phenomenon appears to be the result of the formation of “joints” upon the adsorption of ASMNPs to DNA templates. (b) Nanomaterials are able to inhibit nonspecific amplification products due to incomplete amplification by preferably adsorbing single-stranded incomplete amplification products. (3) Some types of nanomaterials, such as AuNPs, enhance the efficiency and yield of PCR because these types of nanoparticles can adsorb to single-stranded DNA more strongly than to double-stranded DNA. This behavior assists in the rapid and thorough denaturation of double-stranded DNA templates. Therefore, the interaction between the surface of nanoparticles and PCR components is sufficient to explain most of the effects of nanoparticles on PCR.
研究领域:
分子生物学
·
纳米技术
·
生物化学
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DOI
PubMed
高影响因子
BIOTECHNOLOGY AND BIOENGINEERING
·
2021
·Vol. 118
·期 5
·2053-2066
·
IF 4.53
Wei Lin,Tian Tian,Yongzhong Jiang,Erhu Xiong,Debin Zhu,Xiaoming Zhou
Polymerase chain reaction (PCR), a central technology for molecular diagnostics, is highly sensitive but susceptible to the risk of false positives caused by aerosol contamination, especially when an end-point detection mode is applied. Here, we proposed a solution by designing a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 eraser strategy for eliminating potential contamination amplification. The CRISPR/Cas9 engineered eraser is firstly adopted into artpcr reverse-transcription PCR (RT-PCR) system to achieve contamination-free RNA detection. Subsequently, we extended this CRISPR/Cas9 eraser to the PCR system. We engineered conventional PCR primers to enable the amplified products to contain an implanted NGG (protospacer adjacent motif, PAM) site, which is used as a code for specific CRISPR/Cas9 recognition. Pre-incubation of Cas9/sgRNA with PCR mix leads to a selective cleavage of contamination amplicons, thus only the template DNA is amplified. The developed CRISPR/Cas9 eraser, adopted by both RT-PCR and PCR systems, showed high-fidelity detection of SARS-CoV-2 and African swine fever virus with a convenient strip test.
研究领域:
分子生物学
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医学诊断
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基因编辑
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产品关联
DOI
PubMed
高影响因子
FEMS MICROBIOLOGY ECOLOGY
·
2014
·Vol. 87
·期 3
·576-585
·
IF 3.875
Liu Jing,Wang Jia-Kun,Zhu Wen,Pu Yi-Yi,Guan Le-Luo,Liu Jian-Xin
Treponema saccharophilum is a pectinolytic bacterium isolated from the bovine rumen. The abundance of this bacterium has not been well determined, reflecting the lack of a reliable and accurate detection method. To develop a rapid method for monitoring T. saccharophilum, we performed pyrosequencing of genomic DNA isolated from rumen microbiota to explore the 16S rRNA gene sequences of T. saccharophilum candidates. Species-specific primers were designed based on fifteen sequences of partial 16S rRNA genes generated through pyrosequencing with 97% or higher similarity with T. saccharophilumDSM2985 along with sequence from type strain. The relative abundance of T. saccharophilum was quantified in both in vitro and in vivo rumen systems with varied pectin-containing forages using real-time PCR. There was a clear association of T. saccharophilum with alfalfa hay, which contains more pectin than Chinese wild rye hay or corn stover. The relative abundance of T. saccharophilum was as high as 0.58% in vivo, comparable with the population density of other common rumen bacteria. It is recognized that T. saccharophilum plays an important role in pectin digestion in the rumen.
研究领域:
微生物学
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分子生物学
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动物科学
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DOI
PubMed
高影响因子
ANALYST
·
2022
·Vol. 147
·期 4
·625-633
·
IF 5.227
Yan Yu,Ziqing Yu,Xufeng Pan,Lei Xu,Rui Guo,Xiaohua Qian,Feng Shen
Digital polymerase chain reaction (digital PCR) can provide absolute quantification of target nucleic acids with high sensitivity, excellent precision, and superior resolution. Digital PCR has broad applications in both life science research and clinical molecular diagnostics. However, limited by current fluorescence imaging methods, parallel quantification of multiple target molecules in a single digital PCR remains challenging. Here, we present a multiplex digital PCR method using digital melting curve analysis (digital MCA) with a SlipChip microfluidic system. The self-partitioning SlipChip (sp-SlipChip) can generate an array of nanoliter microdroplets with trackable physical positions using a simple loading-and-slipping operation. A fluorescence imaging adaptor and an in situ thermal cycler can be used to perform digital PCR and digital MCA on the sp-SlipChip. The unique signature melting temperature (Tm) designed for amplification products can be used as a fingerprint to further classify the positive amplification partitions into different subgroups. Amplicons with Tm differences as low as 1.5 degrees celsius were clearly separated, and multiple amplicons in the same partition could also be distinguished by digital MCA. We further demonstrated this digital MCA method with simultaneous digital quantification of five common respiratory pathogens, including Staphylococcus aureus, Acinetobacter baumannii, Streptococcus pneumoniae, Hemophilus influenzae, and Klebsiella pneumoniae. Since digital MCA only requires an intercalation dye instead of sequence-specific hydrolysis probes to perform multiplex digital PCR analysis, it can be less expensive and not limited to the number of fluorescence channels.
研究领域:
分子生物学
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临床诊断
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微流控技术
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产品关联
DOI
PubMed
高影响因子
BIOMEDICAL MICRODEVICES
·
2015
·Vol. 17
·期 3
·1-8
·
IF 2.877
Song Qi,Gao Yibo,Zhu Qiangyuan,Tian Qingchang,Yu Bingwen,Song Bofan,Xu Yanan,Yuan Maokai,Ma Congcong,Jin Wei,Zhang Tao,Mu Ying,Jin Qinhan
A nanoliter self-priming compartmentalization (SPC) microfluidic chip suited for the digital polymerase chain reaction (dPCR) analysis in point-of-care testing (POCT) has been developed. This dPCR chip is fabricated of polydimethylsiloxane (PDMS). After the dPCR chip is evacuated, there will be a negative pressure environment in the chip because of the gas solubility of PDMS. The negative pressure environment can provide a self-priming power so that the sample solutions can be sucked into each reaction chamber sequentially. The whole sampling process requires no external power and is valve-free. Channels that contain water are designed around each sample panel to prevent the solvent (water) from evaporating during dPCR process. A glass coverslip is also used as a waterproof layer, which is more convenient and more efficient than other waterproof methods seen in literature. This dPCR chip allows three samples to be amplified at the same time. Each sample is distributed into 1040 reaction chambers, and each chamber is only 2.08 nL. Human β-actin DNA solutions of known concentrations are used as the templates for the dPCR analyses to verify the sensitivity and accuracy of the method. Template DNA solutions diluted to concentrations of 300, 100 and 10 copies/μL are tested and shown that this simple, portable and self-priming dPCR chip can be used at any clinic as a real POCT technique.
研究领域:
分子生物学
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医学检验
·
生物工程
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产品关联
DOI
PubMed
高影响因子
Small
·
2022
·Vol. 18
·期 16
·2107858
·
IF 15.153
Lexiang Zhang,Parvin Rokshana,Yunru Yu,Yuanjin Zhao,Fangfu Ye
Digital PCR (dPCR) surpasses the performance of earlier PCR formats because of highly precise, absolute quantification and other unique merits. A simple thermocycling approach and durable microcarrier are of great value for dPCR advancement and application. Herein, a near-infrared (NIR) controlled thermocycling approach by embedding magnetic graphene oxide (GO) composite into the agarose microcarriers is developed. The core-shell composite is constructed by sequentially encapsulating GO and silica outside the magnetic nanocores. Benefiting from these additives, the resultant composite agarose gains appealing features as light-driven temperature changing, switchable gel–sol phase transforming, biocompatibility, and magnetic traction. By further emulsifying into droplets via the microfluidics method, the influence of typical parameters including material loading amount, laser intensity, and droplet diameter at various ranges is investigated for assembling microcarriers with different responsiveness. Then a paradigm of the NIR program can be easily tailored for PCR thermocycling. Finally, the feasibility of the approach is verified by detecting statistically diluted Klebsiella pneumoniae DNA samples, from 0.1 to 2 copies per drop. It is anticipated that this method has promising prospects for dPCR-based and other temperature-controlled applications.
研究领域:
生物工程
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生物技术
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分子生物学
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分析化学
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产品关联
DOI
PubMed
高影响因子
Tropical Medicine and Infectious Disease
·
2023
·Vol. 8
·期 2
·105
·
IF 2.9
Fengyu Tian,Cong Jin,Shangzhi Ji,Yanqing Tie,Guohao Fan,Ruiqing Zhang,Yehuan Zheng,Xinxin Shen,Xuejun Ma,Zhishan Feng
Human immunodeficiency virus 1 (HIV-1) attacks the immune system, making people susceptible to various diseases, thus increasing their risk of death. Comprehensive detection of major HIV-1 strains circulating in China is vital for effective HIV-1 infection prevention and treatment. HIV-1 nucleic acid detection is considered effective for HIV-1 diagnosis since traditional immunological testing may fail to detect HIV-1 infection during the window period. This work demonstrates a one-pot two-stage amplification assay (RT-RAP), a combination of reverse transcription recombinase (RT- RAA), and quantitative real-time polymerase chain reaction (qRT-PCR). The turn-around time of the assay is only 50 min and can be performed with commonly available laboratory equipment, the qPCR devices. The RT-RAP assay could detect approximately 5 and 14 copies/reaction of HIV-1 DNA and RNA using recombinant plasmids and standard reference strains, respectively. Additionally, we found that the clinical performance of RT-RAP (detected 169 samples out of 170 specimens) was consistent with that of qRT-PCR. The sensitivity and specificity of RT-RAP were 100.00% (99/99) and 98.59% (70/71), respectively, while its positive and negative predictive values were 99.00% (99/100) and 100.00% (70/70), respectively. The total coincidence rate of the RT-RAP was 99.41% (169/170), with a kappa value of 0.988 (p< 0.05). We demonstrated that RT-RAP could rapidly detect the common HIV-1 subtypes commonly circulating in China with comparable sensitivity and specificity to qRT-PCR.Keywords:HIV-1;RT-RAA;qRT-PCR;clinical detection
研究领域:
病毒学
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分子生物学
·
临床诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL BIOCHEMISTRY
·
2022
·
IF 3.191
Junfang Xia,Tong Bu,Pei Jia,Kunyi He,Xin Wang,Xinyu Sun,Li Wang
Polymerase chain reaction (PCR) is one of the most common methods for rapid monitoring of foodborne pathogens; however, it requires purified nucleic acid as a template . Conventional nucleic acid purification is a time-consuming and laborious process. To overcome this, we developed polydopamine nanospheres (PDA NPs)-assisted direct PCR for detecting Escherichia coli O157:H7 ( E. coli O157:H7). PDA NPs significantly enhanced PCR efficiency because of their strong interaction with PCR reagents, including polymerase and primers, thereby enabling regulation of the PCR performance. The optimal concentration and diameter for PDA NPs were 0.10 μg/μL and 504 nm, respectively. The PDA NPs-assisted direct PCR exhibited high sensitivity in E.coli O157:H7 detection. The detection limit of PDA NPs-assisted direct PCR was 6.7 × 10 4 CFU/mL, which was 10-fold lower than that of direct PCR (6.7 × 10 5 CFU/mL). Moreover, the sensor demonstrated excellent selectivity against E. coli O157:H7, with a negative reaction to eight other common pathogens. Most importantly, the PDA NPs-assisted direct PCR detected the order of 10 4−5 CFU/mL E.coli O157:H7 in milk, beef, and watermelon samples. No cultural enrichment was required, with the whole process taking <3 h. Therefore, PDA NPs-assisted direct PCR has tremendous potential in the rapid and sensitive detection of pathogens.
研究领域:
分子生物学
·
食品科学
·
微生物学
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DOI
高影响因子
Biotechnology Journal
·
2021
·Vol. 17
·期 1
·2100335
·
IF 4.677
Shiyu He,Yanzhi Huang,Yingwei Ma,Haoyan Yu,Bo Pang,Xingxing Liu,Caihong Yin,Xiaomu Wang,Yuan Wei,Yuling Tian,Chao Zhao,Kun Xu,Juan Wang,Chunping Lv,Xiuling Song,Minghua Jin
Foodborne pathogen contamination is a major safety issue for many foods and is causing concern worldwide. In this study, a detection system based on magnetic separation, multiplex PCR (MPCR) and capillary electrophoresis (CE) technologies was developed for the simultaneous detection of four foodborne pathogens. Magnetic separation technology is used to rapidly capture pathogenic bacteria in food samples, and then a combination of MPCR and CE can be used to greatly increase detection sensitivity. The detection limit for bacterial DNA reached 10 −5 –10 −7 ng μL −1 and in the analysis of mocked food samples, the assay showed good sensitivity for bacterial detection ranging from 10 1 to 10 5 CFU mL −1 with excellent specificity. Compared to similar detection methodologies, this technique avoids the need for time-consuming enrichment cultures, is more sensitive, and can be used to assay simultaneously four foodborne pathogens. Graphical and Lay Summary Food-borne bacteria contamination is one of the main problems of food safety, which seriously affects the economic development of many countries. In this study, the author achieved rapid separation and enrichment of multiple pathogens in a complex sample matrix through magnetic nano carbon dots, and constructed a rapid detection system for four foodborne pathogens by combining multiplex PCR and capillary electrophoresis technologies. This work shows great potential and application value in food safety monitoring.
研究领域:
食品科学
·
微生物学
·
分子生物学
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产品关联
DOI
PubMed
高影响因子
FOOD CONTROL
·
2025
·
IF 6.3
Kai Chen,Bin Xiao,JianZhi Luo,XueWei Yuan,Juan Zhang,Songxue Wang,Rui Zhang,Nan Wang,LiQiang Liu,AiKe Li,Ailiang Chen
Edible oil adulteration is a significant food safety concern in today's food fraud landscape. DNA-based detection methods emerging as the most reliable approach for identifying adulterated products. However, the processed oil matrix contains only trace amounts of highly degraded DNA, which significantly increases the difficulty of DNA extraction and greatly reduces the success rate of PCR amplification. To address these technical challenges, we designed species-specific primers for amplicons of varying lengths and systematically evaluated their performance in real-time PCR using DNA extracted from soybean, peanut, and rapeseed oils, followed by application in adulterated oil detection. All primer sets demonstrated high specificity, sensitivity and, most importantly, equal amplification efficiency. Results indicated that shorter amplicons were consistently detectable across all three vegetable oils, while longer amplicons were more likely to yield false negatives in real-time PCR reactions. Soybean oil showed CT values of 29 (74 bp) versus 38 (170 bp), peanut oil exhibited a CT value of 31 (54 bp) with no amplification for 86 bp, and rapeseed oil displayed a CT value of 34 (66 bp) with no signal for 101 bp. The use of shorter primers successfully facilitated the detection of adulterated oil.These findings suggest that amplicon length being a critical determinant for reliable DNA-based identification and authentication of edible oils. Thus, designing amplicons within the 50–80 bp range can significantly enhance the success rate of DNA-based identification and authentication of edible oils.
研究领域:
食品科学
·
分子生物学
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）